Bottom Line:
Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases.We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background.These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis.

ABSTRACTBacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.

pone-0039185-g004: The 16 C-terminal amino acid residues of HipB are required for degradation.(A) Degradation of HipB72 in vivo. HipB72 was expressed from a pBRlacitac promoter in BW25113 (KLE905) and its lon::kan derivate (KLE906). Both strains were grown in LB medium, and at an OD600 of 0.3 1 mM IPTG was added. After 1 h of induction, protein synthesis was inhibited by the addition of 100 µg/ml Cam, and samples for Western blots were removed over the course of 30 min. (B) Degradation of HipB 72 in vitro. His6-HipB72 was purified and added to the Lon degradation assay. At least 3 independent experiments were performed to calculate HipB72 turnover.

Mentions:
The HipB dimerization interface is composed of a small hydrophobic core and the β-lid, a two-stranded intermolecular β sheet that is followed by an unstructured 16 amino acid C-terminus (AKNASPESTEQQNLEW) [15]. Proteases typically bind disordered regions of their substrate, thus the unstructured C terminus appears to be an excellent recognition site for protease attack [15]. To test the hypothesis that the 16 residue C-terminal stretch is critical for degradation, we cloned a truncated HipB (HipB72) lacking the last 16 residues of HipB into pBR creating pBRhipB72. We measured the rate of in vivo degradation of HipB72 in wild type and Δlon (KLE905 and KLE906, respectively) (Fig. 4). Interestingly, HipB72 is indeed substantially more stabile (t1/2>200 min) than full length HipB in wild type indicating that the unstructured C terminus of HipB is essential for degradation by Lon protease (Fig. 4A). As expected, full length HipB72 is also stable in Δlon background. We purified the truncated HipB (His6-HipB72) and tested it in the Lon in vitro degradation assay. The effect was also noticeable though less pronounced in vitro. The half-life time of HipB changed from 74 min for full length HipB to 130 min in the mutant (Fig. 4B). To confirm that the unstructured C terminus of HipB is a degradation signal for Lon protease, we fused the C terminus of GFP with the unstructured C-terminal tail of HipB (creating pBRGFP-H, KLE908), and tested whether addition of the carboxy-terminal stretch of HipB (residues 73–88) causes degradation of GFP, which by itself is stable over the time period of the experiment (t1/2>200 min) (Fig. 5). The GFP-HipB tail hybrid was much less stable with a half-life time of ≈53 min confirming that the C-terminus of HipB is critical for rapid proteolysis of HipB.

pone-0039185-g004: The 16 C-terminal amino acid residues of HipB are required for degradation.(A) Degradation of HipB72 in vivo. HipB72 was expressed from a pBRlacitac promoter in BW25113 (KLE905) and its lon::kan derivate (KLE906). Both strains were grown in LB medium, and at an OD600 of 0.3 1 mM IPTG was added. After 1 h of induction, protein synthesis was inhibited by the addition of 100 µg/ml Cam, and samples for Western blots were removed over the course of 30 min. (B) Degradation of HipB 72 in vitro. His6-HipB72 was purified and added to the Lon degradation assay. At least 3 independent experiments were performed to calculate HipB72 turnover.

Mentions:
The HipB dimerization interface is composed of a small hydrophobic core and the β-lid, a two-stranded intermolecular β sheet that is followed by an unstructured 16 amino acid C-terminus (AKNASPESTEQQNLEW) [15]. Proteases typically bind disordered regions of their substrate, thus the unstructured C terminus appears to be an excellent recognition site for protease attack [15]. To test the hypothesis that the 16 residue C-terminal stretch is critical for degradation, we cloned a truncated HipB (HipB72) lacking the last 16 residues of HipB into pBR creating pBRhipB72. We measured the rate of in vivo degradation of HipB72 in wild type and Δlon (KLE905 and KLE906, respectively) (Fig. 4). Interestingly, HipB72 is indeed substantially more stabile (t1/2>200 min) than full length HipB in wild type indicating that the unstructured C terminus of HipB is essential for degradation by Lon protease (Fig. 4A). As expected, full length HipB72 is also stable in Δlon background. We purified the truncated HipB (His6-HipB72) and tested it in the Lon in vitro degradation assay. The effect was also noticeable though less pronounced in vitro. The half-life time of HipB changed from 74 min for full length HipB to 130 min in the mutant (Fig. 4B). To confirm that the unstructured C terminus of HipB is a degradation signal for Lon protease, we fused the C terminus of GFP with the unstructured C-terminal tail of HipB (creating pBRGFP-H, KLE908), and tested whether addition of the carboxy-terminal stretch of HipB (residues 73–88) causes degradation of GFP, which by itself is stable over the time period of the experiment (t1/2>200 min) (Fig. 5). The GFP-HipB tail hybrid was much less stable with a half-life time of ≈53 min confirming that the C-terminus of HipB is critical for rapid proteolysis of HipB.

Bottom Line:
Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases.We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background.These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis.

ABSTRACTBacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.